An electrophoretic separation of nucleic acids in gel matrices usually takes place either in agarose gels or in polyacrylamide gels. The resolving power is determined by the content of agarose in the gel in the case of agarose gels. In contrast, the resolving power of polyacrylamide gels is adjusted by the mixing ratio of acrylamide to bisacrylamide. The detection of nucleic acids in nucleic acid analytics with the aid of gel electrophoresis is based on the fact that fluorescent DNA binding dyes permanently bind non-covalently to nucleic acids and in their bound form enable nucleic acids to be located in the gel matrix after excitation with light of a suitable wavelength.
In classical gel electrophoresis the intercalating dye ethidium bromide was used for this purpose. However, due to the toxicity of ethidium bromide, alternative DNA binding dyes such as for example SYBR Green or SYBR Gold (Molecular Probes, Inc.) have been used for several years, the binding properties of which are not or not exclusively based on the principle of DNA intercalation.
In contrast to ethidium bromide, these dyes can also be used to detect PCR amplificates in real time PCR or qPCR since they do not substantially inhibit the PCR reaction (U.S. Pat. No. 6,569,627). There are other dyes apart from SYBR Green and SYBR Gold which can also be used in real time PCR (Gudnason, H., et al., Nucleic Acids Research 35(19) (2007) e127).
Furthermore, certain dyes exist for real time PCR applications which are especially suitable for thermal melting curve analyses such as the LIGHTCYCLER (Roche Diagnostics GmbH) 480 RO 27 dye (Roche Applied Science Cat. No.: 04 909 640 001). They are grouped together as HRM (high resolution melting) fluorescent dyes and are characterized in that they can be used in higher concentrations in the PCR without inhibiting the PCR reaction.
Capillary gel electrophoresis is also an important method for analyzing PCR products, for analyzing fragments from the restriction digestion, for mutation analytics and DNA sequencing due to its speed, the ability to automate and its high resolving power. LIF (laser induced fluorescence) detection plays an important role in this connection in order to make it possible to detect and quantify DNA in a highly sensitive manner and in a high dilution. In addition to the covalent coupling of fluorescent dyes to the DNA (by means of fluorescent labelled primers during DNA sequencing), the use of dsDNA intercalators plays a major role as a non-covalent method which enables DNA to be detected fluorimetrically and simply by means of stable dye-DNA complexes. The same intercalators are used for this as for the detection of DNA in gel electrophoresis (Sang, F., et al., J. Sep. Sci. 29 (2006)1275-1280).
The dyes used for electrophoresis are either admixed with the gel preparation before polymerization in the case of ethidium bromide, or the gel is stained after completion of the gel electrophoresis with the aid of an aqueous dye solution containing ethidium bromide or another dye. Furthermore, it has also been described that SYBR Green I can be added to the sample containing the nucleic acid before loading the gel, provided the nucleic acid is incubated with the dye for a further 15 minutes before being applied to the gel (Karlsen, F., et al., Journal of Virol. Methods 55 (1995) 153-156, and Jin, X., et al., FASEB J. 10 A1128 (1996) abstract # 751).
The staining methods for the gel electrophoretic analysis of nucleic acids known from the prior art thus have the disadvantage that the addition before polymerization only takes place with the mutagenic substance ethidium bromide, staining in a dye solution is time-consuming and laborious and requires large amounts of dye, or the addition of the dye immediately before loading the gel requires a longer incubation period.